Project Summary: The human mitochondrial genome (MG) is prone to genetic alterations, which lead to somatic mutations, changes in mitochondrial DNA (mtDNA) copy number and altered expression of respiratory chain subunits. This is in part due to the fact that mtDNA has a limited ability to repair itself when it is damaged. Although a buildup of somatic mutations in mtDNA has been associated with increased cancer risk, the role of mtDNA alterations in malignant transformation of lung adenocarcinoma (LA) has not been addressed. Therefore, a clear understanding of changes in mtDNA along the pathway of lung tumorigenesis is critical for identifying molecular biomarkers related to carcinogenesis and tumor progression. Like all solid tumors, LA is thought to be initiated and to progress through a series of genetic alterations, including changes in mtDNA. Lungs resected for primary adenocarcinomas often harbor minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and ultimately fully invasive adenocarcinoma. To delineate mitochondrial clonal heterogeneity as a function of tumor progression we will use novel high- throughput-PCR-based enrichment technology combined with next generation sequencing (NGS) to assess mtDNA alterations in samples isolated from AAH lesions collected from patients diagnosed with primary invasive adenocarcinoma or from different zones of histologic progression within the same AIS and MIA. In this way, we will gain a unique understanding of the mitochondrial heterogeneity of early lesions and the potential role of these clonal events in the progression of early glandular neoplasms. Next, we will sequence LA that were detected through spiral CT screening approach and interval cancers, which likely indicate their rapidly progressing phenotypes. Finally, we will validate mtDNA-derived mutations in paired plasma samples using a sensitive and robust digital PCR approach, which provides a novel opportunity for noninvasive early lung cancer detection. In summary, this proposal will identify mtDNA-derived somatic mutations along the progression of LA and will develop biomarkers that will allow us to better predict the fate of early lesions non-invasively.